Blood is a complex dispersion of colloidal particles (e.g., red and white blood cells, proteins, etc.) in an acellular continuous plasma phase. The components of the disperse phase of blood provide most of the biological functions including oxygen transport to tissues, hemostasis, host defense, transport of nutrients and hormones, and the removal of metabolic wastes. Although all of the functions of blood are important, the quintessential function of blood is the delivery of oxygen. Oxygen deprivation or ischemia quickly leads to irreversible degradation of cells, tissues and organs.
Currently, human blood is the agent of choice for acutely and chronically anemic patients. However, donated blood is not without its risks. Donated blood may be contaminated by a large number of pathogens, such as the human immunodeficiency virus (HIV), hepatitis or prions which cause variant Creutzfeldt-Jakob disease. Transfusion of allogeneic blood can also result in immunosuppression which has been linked to increased recurrence of cancer and incidents or postoperative infections in surgical patients. Blumberg et al., Blood, 66 Supp. 1, 274a (1966); Maetani et al., Ann. Surg, 203, 275 (1986).
Although the public's perception of risk focuses on risk of infection, clerical errors leading to mistransfusion are the most common causes of serious morbidity and mortality related to blood transfusion. The reported incidence of administration of RBCs to other than the intended recipient is 1 in 19,000; however, an actual incidence of 1 in 400 units was found in a prospective study of transfusion errors. American Association of Blood Banks, Noninfectious Serious Hazards of Transfusion, Association Bulletin, 01-4 (2001). During storage of blood, biochemical and morphologic changes occur in the blood that results in irreversible RBC damage and reduced post transfusion survival. This so-called “storage lesion” causes depletion of 2,3-diphosphoglyceric acid (2,3-DPG), resulting in less efficient oxygen transport by hemoglobin until naturally repleted, a process taking many hours. Valeri et. al, “Restoration in vivo erythrocyte adenosine triphosphate, 2,3 diphosphoglycerate, potassium ion and sodium ion concentrations following transfusion of acid-citrate-dextrose-stored human red blood cells.” J. Lab Clin Med., 73:722-33 (1969). Stored blood also contains bioreactive substances such as cytokines, which are implicated as a primary cause of nonhemolytic febrile transfusion reactions.
While disease transmission and clerical error remain serious issues for blood transfusion, the availability of donor blood has recently emerged as a significant concern on an international level. Concern over blood shortages appears to be driven in part by recent guidelines to defer blood donors who have spent extended periods of time in Europe, due to the potential risk of contracting variant Creutzfeldt-Jakob Disease (vCJD) from consumption of infected beef. In addition, a decreasing blood donor pool (due to an aging population and greater donor restrictions) and an increasing demand for blood for major surgical procedures in a larger number of elderly patients are resulting in frequent regional shortages causing cancellations or delays in elective surgery. Donor blood, when administered, is often old and must be typed and crossmatched for each patient, a process which can result in dangerous transfusion delays. Blood must also be refrigerated and has a shelf life of approximately 42 days, making it unavailable in many critical instances such as trauma situations in remote areas. Shortage of stored blood is a significant worldwide problem with blood stocks only having a several day supply in many cases, making coping with a disaster very difficult.
The search for therapeutic agents to replace the oxygen transport function of blood continues to be a high priority throughout the world. The goal is to find a replacement to donor blood which is pathogen free, stable, storable for long periods of time, affordable, and a universal donor product that would be immediately available when and where it is needed. Currently, there are two diverse approaches being undertaken in blood substitutes, purified hemoglobin derivatives and fluorochemical emulsions.
1. Purified Hemoglobin Based Oxygen Delivery
Efforts have been underway for some time to produce hemoglobin based oxygen carriers. Hemoglobin is a tetrameric protein of approximately 64,000 daltons which carries oxygen throughout the body. Hemoglobin is composed of four subunit polypeptide chains of about 140 amino acids each. Each chain has a molecular mass of 16,100 daltons and carries a tetrapyrrole iron-containing prosthetic group, heme, which can bind to one molecule of oxygen. In humans there are several different types of hemoglobins and all types contain four subunits. The differences in hemoglobin subtypes in humans are limited to the primary structure (amino acid sequence) of globin.
Blood transports oxygen bound to hemoglobin within the red blood cells which distribute oxygen throughout the tissues. Consequently, hemoglobin's ability to bind and release oxygen has made it an attractive subject for a blood substitute. However, hemoglobin is an inherently unstable molecule. Outside of its red blood cell environment, the hemoglobin molecule rapidly dissociates into dimers composed of an alpha and a beta subunit which are rapidly removed from circulation by the kidneys. Thus, development of a suitable stroma free hemoglobin molecule depends on the development of a stable, functional tetramer of hemoglobin which does not dissociate into dimers upon infusion. Another problem of hemoglobin outside of its red blood cell environment is caused by its having a high oxygen affinity (due to the lack of its normal allosteric effector, 2,3-diphosphoglycerate normally present in red blood cells) and the potential in high concentrations to cause renal tubular obstruction and consequent renal failure. Thus, in order to be an effective oxygen carrier in a cell-free state or in solution, hemoglobin must be chemically modified to avoid the problem of disassociation and oxygen affinity. Secondary goals are to produce a high yield of product in a cost effective manner.
Efforts to solve this problem have been undertaken in several novel ways including chemically binding the hemoglobin protein subunits together to prevent dissociation (e.g., by the binding of pyridoxal phosphate to the hemoglobin molecule or various other cross linking strategies) and the production of recombinant hemoglobin. Problems with chemically altering hemoglobin include ensuring an adequate supply of the raw hemoglobin material such as human blood which is in short supply. The use of hemoglobin obtained from other mammals, such as bovine derived hemoglobin, is a concern due to the bovine spongiform encephalitis virus and other pathogens. One alternative is recombinant hemoglobin. The problem with recombinant hemoglobin is its low yield and therefore its high cost of production. There are also purity concerns as endotoxin contamination is often a problem with Escherichia coli products
Another problem of artificial hemoglobin is its binding of other free gases. Free hemoglobin avidly binds nitric oxide. It is unknown whether this in vivo binding is of clinical significance, although binding of nitric oxide has been implicated as the cause of hypertension commonly seen following hemoglobin infusion. It remains to be determined what effects stroma free hemoglobin has on regional autoregulation of blood flow, and whether the hypertension associated with hemoglobin infusion has pathophysiologic consequences. At present, little data are available in large animal or clinical studies utilizing these compounds to elucidate the importance of this phenomenon.
2. Fluorocarbon Emulsions
Fluorochemicals are molecules comprised of fluorine atoms. The term fluorochemical or fluorocarbon is contrasted with the term perfluorochemicals (“PFCs”), which are chemically inert synthetic molecules consisting primarily of carbon and fluorine atoms (i.e., no hydrogen atoms). Liquid PFCs and fluorocarbons are generally clear, colorless, and practically odorless, and possess the intrinsic ability to physically dissolve significant quantities of many gases, including oxygen, carbon dioxide and nitrogen. Because PFCs are hydrophobic, they are not miscible with water and therefore must be emulsified with a surfactant (e.g., phospholipid) to create an aqueous-based PFC emulsion for intravenous use. By mixing the PFC, surfactant and an aqueous buffer under high shear (e.g., homogenization) conditions, tiny submicron-sized droplets are formed in the aqueous media. The PFC droplets are surrounded by a monolayer of surfactant in which the hydrophobic lipid end of the surfactant molecules orient themselves into the PFC-containing core while the hydrophilic phosphate-containing polar head groups form the outer surface of the droplet where they are exposed to the aqueous environment.
PFCs and fluorocarbons do not deliver oxygen to tissues in the same manner as hemoglobin. Oxygen is highly soluble in fluorocarbon compounds, which, after intravascular injection, are present in the plasma phase of blood. Thus the contribution of fluorochemicals to oxygen delivery is due to their ability to increase O2 carried in the plasma compartment. Although the absolute amount of O2 carried by fluorocarbon compounds is relatively small, even at high inspired Fractional Oxygen Concentrations (FiO2), a very high percentage of the transported O2 is released at the tissues resulting in O2 extraction from the fluorocarbon phase, often in excess of 90%.
PFC emulsions augment the total oxygen content of the blood by increasing the dissolved oxygen carried within the plasma compartment in an amount linearly proportional to the oxygen partial pressure (PO2). At elevated levels of PO2 (patient inspiring high concentrations of oxygen), oxygen within the PFC emulsion is more readily available to tissues than the oxygen bound to hemoglobin in red blood cells (RBCs). This is because oxygen from PFC emulsions load and offload linearly, whereas oxygen from red blood cells chemically bind and release according to the S-shaped oxyhemoglobin disassociation curve. Extraction of O2 from hemoglobin at the tissues under normal circumstances is in the range of 20-25% and is lower overall than when fluorocarbon compounds are in the circulation delivering fluorocarbon-dissolved O2 to the tissues.
Hemoglobin is nearly saturated at atmospheric oxygen levels and its oxygen content cannot be enhanced by any significant amount by increasing the inspired oxygen concentration. The extraction ratio for PFC emulsions is about 60% as compared to about 20-25% for hemoglobin under ambient conditions. When high concentrations of oxygen are inspired oxygen extraction from PFC emulsions reaches 90% or more. Consequently, when a fluorocarbon or PFC emulsion is present in the blood, the fluorocarbon or PFC emulsion will always release its oxygen load first, thus conserving the oxygen bound to hemoglobin. Numerous in vivo and in vitro studies support the efficacy of PFC emulsions for enhancement of oxygen delivery and maintenance or improvement of systemic and tissue oxygenation during surgical procedures.
Since the early 1960s, a number of different research efforts have attempted to develop a stable fluorocarbon emulsion for use as an intravascular oxygen therapeutic. The first commercial development of an injectable fluorocarbon emulsion was achieved approximately 30 years ago by the Green Cross Corporation (Osaka, Japan) with the production of FLUOSOL, a 20% w/v PFC emulsion comprising 14% w/v perfluorodecalin and 6% w/v perfluorotripropylamine, emulsified primarily with a synthetic poloxamer, Pluronic F-68 and a small amount of egg yolk lecithin. Limitations of this first generation product included the need for frozen storage of the stem emulsion, the need to thaw the emulsion and subsequently mix with two annex solutions prior to use. Short product stability after reconstitution (8 hours) and significant side effects (e.g., severe alternate pathway complement activation) were big problems for FLUOSOL which were primarily caused by the synthetic Pluronic surfactant.
FLUOSOL was tested extensively in severely anemic and actively bleeding Jehovah's Witness patients, and clearly demonstrated the ability to deliver oxygen. Tremper et al., “The preoperative treatment of severely anemic patients with a perfluorochemical oxygen-transporting fluid, Fluosol-DA.”; N Engl J Med 307; 277-83 (1982); Gould et. al, “Fluosol-DA as a red-cell substitute in acute anemia.” N Engl J Med 314: 1653-6 (1986).
FLUOSOL, however, did not receive FDA approval for this large-volume “blood substitute” indication because the temporary oxygenation benefit did not significantly improve mortality outcome in these bleeding and extremely anemic patients who refused blood transfusions due to their religious beliefs. Subsequent efforts to develop FLUOSOL focused on using it as an adjunct to coronary artery balloon angioplasty (PTCA) procedures, i.e., as an oxygen-carrying low-viscosity fluid that could be perfused through the PTCA catheter to oxygenate the distal myocardium during prolonged balloon inflation. Efficacy of FLUOSOL treatment during PTCA was clearly demonstrated based on attenuating myocardial ischemia, maintaining ventricular function (improved cardiac output) during balloon inflation and decreasing wall motion artifacts (i.e., decreased ST segment elevations and improved left ventricular ejection fraction). Bell et al., “Does intracoronary infusion of Fluosol-DA 20% prevent left ventricular diastolic dysfunction during coronary balloon angioplasty?” J Amer Coll Cardiol 16: 959-66 (1990); Cowley et al., “Perfluorochemical perfusion during coronary angioplasty in unstable and high-risk patients.” Circulation 81 (Supp IV): IV-27-34 (1990). These data formed the basis for FLUOSOL's marketing approval in the United States, which was granted by the FDA in December 1989. To date, FLUOSOL represents the only synthetic oxygen therapeutic approved by the FDA.
Additional clinical testing during the late 1980s and early 1990s occurred in cancer patients using FLUOSOL as an adjunct to primary radiation. These studies included patients with advanced head and neck malignancies, anaplastic astrocytomas, carcinoma of the lung and glioblastomas multiforme. In the early 1990s, FLUOSOL was also tested in a large multicenter clinical study (TAMI-9) in 430 patients with acute myocardial infarction, to assess the safety and efficacy of FLUOSOL as an adjunct reperfusion therapy following treatment with a thrombolytic agent (tissue-type plasminogen activator). A trend was observed for lower mean infarct size and less recurrent ischemia in the FLUOSOL-treated group. Unfortunately, significant improvement was not seen in global ejection fraction, regional wall motion or left ventricular ejection fraction. Due to the large volumes of FLUOSOL administered (15 mL/kg), a tendency for transient congestive heart failure and pulmonary edema was observed, which likely attenuated the oxygenation efficacy benefit of the FLUOSOL treatment.
Despite these efforts to develop secondary indications, Green Cross stopped manufacturing FLUOSOL in early 1994, primarily due to poor sales in the PTCA market (since autoperfusion catheters had since entered the market which now allowed blood to be perfused through the lumen of the catheter during balloon inflation). In addition, data had become available that prolonged balloon inflation times did not correlate with a decrease in the rate of coronary restenosis post-PTCA. Nevertheless, the FDA approval of FLUOSOL represented a very significant milestone in the development of oxygen therapeutics. This approval demonstrated that a PFC-based emulsion was both safe and efficacious as a temporary intravascular oxygen carrier to ameliorate hypoxia in ischemic tissues in patients.
During the last 10-15 years, commercial research efforts have resulted in the development of second-generation fluorocarbon emulsions with improved product characteristics compared to the first generation dilute formulations. The most successful of these efforts focused on more versatile linear chain fluorocarbon compounds (instead of cyclic fluorocarbon molecules) that have even slightly higher oxygen solubility. An additional improvement in these second-generation fluorocarbon emulsions involved the use of lecithin, i.e., egg yolk phospholipid (EYP), as the surfactant. EYP has been used for years to make parenteral products such as INTRALIPID (i.e., triglyceride-based fat emulsions for intravenous feeding of patients who cannot ingest food), and is much more biocompatible than the synthetic Pluronic-based surfactants used previously.
Another development effort to produce a fluorocarbon-based oxygen carrier has been on-going for many years in Russia, but relatively little information is available in the English literature for this product. PERFTORAN, originally developed at the Institute of Theoretical and Experimental Biophysics (Puschino, Russia), is a 20% w/v emulsion consisting of 14% w/v perfluorodecalin and 6% w/v perfluoro-n-methylcyclohexylpiperidine emulsified with a synthetic poloxamer (Proxanol) similar to Pluronic F-68. Average particle size in the PERFTORAN emulsion is <0.2 μm. However, the emulsion must be stored frozen (for up to 3 years), but after thawing it can be kept refrigerated for only 2 weeks. Perftoran has apparently been evaluated in more than 500 patients across a wide variety of different clinical and medical indications including battlefield use on soldiers suffering from traumatic blood loss. Vorobyev et al., “Perfluorocarbon emulsion Perftoran—The plasma substitute with gas transporting function.” Artif Cells Blood Subst Immob Biotech 24: 453 (1996).
In Russia, PERFTORAN was approved in 1999 for human use. The indication statement for PERFTORAN claims that it is to be used “as a blood substitute preparation with gas transporting function in case of shock, blood losses, multiple trauma, burning of large surface of skin, conditions of apparent death, as well as in transplantology.” It has also been used clinically in cardiopulmonary bypass, in regional perfusion to treat limb ischemia, and for severe alcohol intoxication. It is noteworthy, however, that no studies with PERFTORAN have ever been performed outside of Russia, reportedly due to their inability to manufacture the product according to cGMP guidelines.
Synthetic Blood International, Inc. (SBI) has been working for many years on PFCs, based on the early pioneering work of Leland Clark Jr. The initial focus of this company was to develop an implantable glucose biosensor for diabetics, and to utilize PFCs for liquid ventillation (Fluorovent). Only in the past few years, has SBI formulated a concentrated 60% w/v PFC emulsion apparently based on a custom-synthesized C10F20 proprietary PFC molecule. This compound is claimed to possess physical properties that are favorable for making biocompatible PFC emulsions for in vivo use. However, their proprietary compound is likely difficult to synthesize at very high levels of purity, and is probably expensive to produce since it requires a custom synthesis. Also, the tissue residence time will likely be longer than other more commonly used PFCs, such as pefluorodecalin or perflubron. In February 2003, SBI announced the filing of their IND for OXYCYTE, and received FDA approval to start a dose-escalation Phase 1 study in April 2003.
In the early 1990s, Hemagen/PFC (St. Louis, Mo.) attempted to use a custom-synthesized fluorochemical, perfluorodichlorooctane (C8F16Cl2; PFDCO) when they attempted to develop OXYFLUOR as a concentrated emulsion. Kaufman R J. “Clinical development of perfluorocarbon-based emulsions as red cell substitutes.” Blood Substitutes: Physiological Basis of Efficacy. Boston: Birkhauser, 52-75 (1996). Hemagen/PFC attempted a novel approach by adding oil to the emulsion in order to decrease the particle size of their OXYFLUOR emulsion and thereby increase product stability. The resulting OXYFLUOR emulsion was a 3-phase formulation based on combining PFDCO with triglyceride (safflower oil) and EYP as the only surfactant (78% w/v (40% v/v) fluorocarbon emulsion). The OXYFLUOR emulsion had an average particle size of 0.22-0.25 μm, but actually consisted of two populations of different particles; i.e., small oil (triglyceride) droplets and larger PFC-containing droplets. As a result, the side effect profile in humans was more pronounced, causing significant febrile reactions and flu-like symptoms due to the acute phase reaction triggered by these larger sized emulsion particles. Kaufman R. “The results of a Phase 1 clinical trial of a 40% v/v % emulsion of HM351 (Oxyfluor™) in healthy human volunteers.” Artif Cells Blood Subst Immob Biotech; 22: A112 (1994).
Hemagen was pursuing an indication to ameliorate the cognitive dysfunction that is commonly encountered post-bypass, which is presumably due to local cerebral ischemia caused in part, by gaseous microemboli generated during cardiopulmonary bypass. An early stage Phase 2a safety study in cardiopulmonary bypass was initiated, but all patients had to be pretreated with dexamethasone to suppress the acute side effect profile. This ultimately led to the demise of these clinical studies, and development was ultimately stopped.
Despite the problems of various fluorocarbon emulsions as described, depending on the choice of specific PFC and surfactant, it is possible to make stable fluorocarbon-in-water emulsions for in vivo use in humans with exceptionally small particles (median diameter<0.2 μm) which remain stable over time and are biocompatible. However, the selection of a particular fluorocarbon(s) with the appropriate physical and chemical properties, in the proper concentrations and amounts, and the proper surfactant, is critical in determining how safe (i.e., biocompatible) a fluorocarbon emulsion will be in vivo, and how readily the fluorocarbon molecules will be eliminated from the body. Inadequate purity of a fluorocarbon, that contains other fluorocarbon compounds or partially fluorinated contaminants, can adversely affect the safety of the fluorocarbon emulsion and often results in toxicity. Fluorocarbon characteristics like molecular weight, lipid solubility, and vapor pressure are all major factors that directly influence the behavior of fluorocarbons in vivo. In addition, these fluorocarbon properties and the selection of an appropriate surfactant for use with the fluorocarbons, with the right chemical and physical properties, ultimately determines the intrinsic stability and shelf life of the final fluorocarbon emulsion formulation.
Pharmacokinetics of Intravenously Administered Fluorocarbon Emulsions
The pharmacokinetics of intravenously administered fluorochemical emulsions may be described by the four compartment illustration in FIG. 1. Emulsion droplets are cleared from the circulation through phagocytosis by circulating monocytes or tissue resident macrophages of the reticuloendothelial system (“RES”). Circulating monocytes pass through the pulmonary circulation and phagocytize emulsion droplets in the circulation, then migrate to the alveolar space where the fluorochemical is transported across the blood/air interface and eliminated in expired air. The rate constant for this process, k10, has been shown to be very small indicating that little fluorocarbon is removed by this pathway. The dominant blood removal mechanism involves uptake and phagocytosis of the droplets by the cells of the RES. Approximately 80% or more of the administered dose is found in the organs of the RES after removal from the blood, principally the liver and spleen.
Chemically and biologically perfluorocarbons are inert and thus no metabolism is observed for perfluorochemicals. Following phagocytosis, intracellular fluorochemical is removed from RES cells (k24) by incorporation in circulating lipid carriers (i.e., chilomicrons and lipoproteins). At this point the fluorochemical can be eliminated in expired air (k40), partitioned into adipose tissue (k43) or returned to the RES (k42). The ultimate removal depends on the magnitude of the rate constants which populate and depopulate compartment 4. The rate determining step, k24, is controlled by the mass transfer of fluorochemical into the lipid carriers, a process which depends critically upon the lipid solubility of the fluorochemical. R. E. Moore and L. C. Clark in R. Frey et al., “Oxygen Carrying Colloidal Blood Substitutes: 5th Int. Symp. On Perfluorochemical Blood Substitutes, Munich, Germany,” pg. 50 (1982). The concentration of fluorochemical found in compartment 3 (adipose tissue) is also dependent on the lipophilic nature of the fluorochemical. Because adipose is poorly perfused, the removal will be slow compared to the RES which has a rich circulation. The redistribution into adipose leads to a decrease in the whole body removal rate (k40). For a typical fluorochemical (e.g., perfluorooctyl bromide), the rate constants have the following magnitude in units of hr−1: k10≈0.000, k12≈0.04, k24≈0.006, k42≈0.002, k34≈0.002, k40≈0.07.
The nature of the fluorochemical can have profound effects on the observed pharmokinetics. As discussed, k24 depends primarily on the lipophilicity of the fluorochemical. Indeed, significant differences in k24 have been observed for the two fluorochemical components of the fluorocarbon emulsion FLUSOSOL, a fluorocarbon emulsion approved by the United States Food and Drug Administration to deliver oxygen to tissues during balloon angioplasty procedures (The Green Cross Corp., Osaka, Japan). The rate constants for RES elimination (k24) are equal to 0.10 and 0.011 day−1 for F-decalin and F-tripropylamine, respectively, and body clearances are much slower than for perfluorooctyl bromide.
The intravascular persistence of the fluorochemical is also an important factor in its biocompatibility since it is directly proportional to the efficacy of an emulsion product designed for use in “blood substitute” applications. The magnitude of k12 (uptake of particles by the RES) depends critically on the total dose of fluorochemical, the emulsion droplet size, and possibly the binding of specific opsonins or dysopsonins which promote droplet recognition by the RES, or alternatively give “stealth-like” characteristics to the emulsion droplets. In fluorochemical emulsions containing two phase disperse phase components, significant partitioning of the fluorochemical components is observed between different sized droplets due to molecular diffusion of the more water soluble components through the continuous phase. This leads to a situation where the more water soluble fluorochemical is concentrated in the larger droplets, while the smaller droplets are enriched in the slower diffusing insoluble component. Since different sized droplets have different fluorochemical quantities, it is possible to imagine a situation where the individual rate constants for removal from the blood might differ somewhat, especially as the larger sized droplets are selectively removed by the RES.
More direct evidence of the importance of fluorochemical lipophilicity in the removal process comes from the work of Obraztsov et al. who proposed a two step removal mechanism of perfluorochemicals. According to the model, the first step is the molecular diffusion of perfluorochemicals through the cytoplasm of the RES cells to the blood stream. This process occurs in a time span of minutes to hours. The second (rate-determining) step involves the mass transfer of fluorochemical from the RES organs to the lungs by the lipid carriers, a process which depends critically on PFC lipophilicity. In an elegant experiment, Obraztsov found that the organ retention time of perfluorochemicals can be decreased significantly by post-intravenous injection of a lipid emulsion given following PFC emulsion administration. The lipid emulsion is therefore able to provide a lipid sink in the blood to remove the PFC from the organs and carry it to the lungs. Obratzsov, V. V et. al, J. Fluorine Chem. 54, 376 (1991).
Stability of Fluorocarbon Emulsions
Submicron fluorochemical emulsions designed for oxygen transport are thermodynamically unstable. The primary mechanism of irreversible droplet coarsening is Ostwald ripening. J. G. Reiss, Colloids Surfaces, 84, 33 (1994). Ostwald ripening occurs as a consequence of the Kelvin effect, whereby small differences in surface tensions between different size droplets leads to growth of the larger droplets and shrinkage of the smaller ones with time. Mass transfer between droplets occurs via molecular diffusion of the disperse phase through the continuous phase. Ostwald ripening can occur not only after the emulsion is made during storage but also during the manufacturing process. To counteract emulsion coarsening via Ostwald ripening, Haguchi and Misra proposed the addition of a higher molecular weight second disperse phase component which is less insoluble in the continuous phase. Higuchi et al., J. Pharm. Sci, 51, 459 (1962). In this case, significant partitioning of the two disperse phase components between different droplets occurs, with the component having low water solubility being concentrated in the smaller droplets.
During Ostwald ripening in two component phase systems, equilibrium is established when the difference in chemical potential between different sized droplets, which results from capillary effects, is balanced by the difference in chemical potential resulting from partitioning of the two components (similar to Raoult's law for vapor/liquid equilibria).
The initial droplet size and distribution, droplet stability, and ultimately many of the observed side-effects found for fluorochemical emulsions, depend critically upon reducing Ostwald ripening. The physical stability of fluorocarbon emulsions, therefore, depends critically on the nature of the dispersed fluorocarbon phase.
The kinetics of droplet growth via molecular diffusion is most often described in terms of the Lifshitz-Slezov-Wagner (“LSW”) theory. LSW theory relates that for a single component disperse phase, the cube of the mean radius increases linearly with time at a rate, {acute over (ω)}.{acute over (ω)}=d/dt(a)3=8γVCaD/9RT where a is the radius, γ is the interfacial tension, V is the molecular volume, Ca is the water solubility, D is the diffusion coefficient, R is the molar gas constant, and T, the absolute temperature. Of particular importance for fluorocarbon emulsion stability is the water solubility term, a parameter which depends critically on the molecular weight of the fluorocarbon. In general, fluorocarbons with higher molecular weights exhibit reduced water solubility, and hence, greater emulsion stability. However, fluorocarbon emulsions destined as injectable oxygen carriers (i.e. blood substitutes) must also be biocompatible. Of particular importance is the half-life of the fluorocarbon in the organs of the RES. Although higher molecular weight fluorocarbons exhibit enhanced emulsion stability, they are also retained in the RES for extremely long periods of time.
Ostwald ripening may be decreased by inclusion of a secondary fluorochemical of higher molecular weight and lesser water solubility or a fluorinated surfactant which significantly reduces the interfacial tension at the fluorocarbon/water interface. Unfortunately, the addition of less water soluble secondary fluorocarbons to a formulation leads to increases in organ retention, a very serious side effect, since the fluorochemical's solubility in circulating lipid carriers will also be reduced. Fluorinated surfactants have proven to have serious toxicity issues making them risky as an emulsion stabilizer. In order to overcome the emulsion stability/organ retention dilemma, a secondary fluorocarbon must be chosen which provides the required emulsion stability/organ retention characteristics, is biocompatible with short organ retention times, and added in the smallest quantities possible while obtaining the needed benefit in stability. For this to occur, the chosen fluorochemical should be lipophilic (e.g., perfluorodecyl bromide). Nonlipophilic compounds (e.g. F-tripropylamine and F—N-methylcyclohexylpeperidine) are excreted too slowly and may not effectively stabilize fluorochemical emulsions.
Some representative primary and secondary fluorocarbons are included in the list below.
1. Primary Fluorocarbon
The primary fluorocarbon is selected for its short organ retention time and biocompatibility. In general, the half life in organs is preferably less than about 4 weeks, more preferably less than about 2 or 3 weeks, and most preferably 7 days or less. The molecular weight is from about 460 to about 550 daltons.
Such possible primary fluorocarbons include bis(F-alkyl)ethenes such as C4F9CH═CHC4F9 (“F-44E”), 1-CF3CF9CH═CHC6F13 (“F-i36E”), and cyclic fluorocarbons, such as C10F18 (F-decalin, perfluorodecalin or FDC); F-adamantane (FA); perfluoroindane; F-methyladamantane (FMA); F-1,3-dimethyladamantane (FDMA); perfluoro-2,2,4,4-tetramethylpentane; F-di- or F-tri-methylbicyclo[3,3,1]nonane (nonane); C7-12 perfluorinated amines, such as F-tripropylamine, F-4-methyloctahydroquinolizine (FMOQ), F-n-methyl-decahydroisoquinoline (FMIQ), F-n-methyldecahydroquinoline (FHQ), F-n-cyclohexylpyrrolidine (FCHP), and F-2-butyltetrahydrofuran (FC-75 or RM101).
Other examples of primary fluorocarbons include brominated perfluorocarbons, such as perfluorooctyl bromide (C8F17Br, USAN perflubron), 1-bromopentadecafluoroheptane (C7F15Br), and 1-bromotridecafluorohexane (C6F13Br, also known as perfluorohexyl bromide or PFHB. Other brominated fluorocarbons are disclosed in U.S. Pat. Nos. 3,975,512 and 4,987,154 to Long and U.S. Pat. Nos. 5,628,930 and 5,635,538, which are hereby incorporated by reference in their entirities.
Also contemplated are fluorocarbons having other nonfluorine substituents, such as 1-chloro-heptadecafluorooctane (C8F17Cl, also referred to as perfluorooctyl chloride or PFOCl); perfluorooctyl hydride, and similar compounds having different numbers of carbon atoms.
Additional first fluorocarbons contemplated in accordance with this invention include perfluoroalkylated ethers, halogenated ethers (especially brominated ethers), or polyethers, such as (CF3)2CFO(CF2CF2)2OCF(CF3)2; (C4F9)2O. Further, fluorocarbon-hydrocarbon compounds may be used, such as, for example compounds having the general formula CnF2+1—Cn′H2n′+1; CnF2n+1OCn′H2n′+1 or CnF2n+1CH═CHCn′H2n′+1, wherein n and n′ are the same or different and are from about 1 to about 10 (so long as the compound is a liquid at room temperature). Such compounds, for example, include C8F17C2H5 and C6F13CH═CHC6H13.
Other possible fluorocarbons for use as the primary fluorocarbon include perfluoroamines, terminally substituted linear aliphatic perfluorocarbons having the general structure:                CnF2n+1R, wherein n is an integer from 6 to 8 and R comprises a lipophilic moiety selected from the group of Br, Cl, I, CH3, or a saturated or unsaturated hydrocarbon of 2 or 3 carbon atoms,bis(F-alkyl)ethenes having the general structure:        
CnF2n+1—CH═CH—Cn′F2n′+1, wherein the sum of n and n′ equals 6 to 10, and perfluoroethers having the general structure:
CnF2n+1—O—Cn, F2n+1, wherein the sum of n and n′ equals 6 to 9.
In addition, fluorocarbons selected from the general groups of perfluorocycloalkanes or perfluoroalkyl-cycloalkanes, perfluoroalkyl saturated heterocyclic compounds, or perfluorotertiary amines may be suitably utilized as the first fluorocarbon. See generally Schweighart, U.S. Pat. No. 4,866,096, which is hereby incorporated by reference in its entirety.
It will be appreciated that esters, thioethers, and other variously modified mixed fluorocarbon-hydrocarbon compounds, including isomers, are also encompassed within the broad definition of fluorocarbon materials suitable for use as the first fluorocarbon of the present invention. Other suitable mixtures of fluorocarbons are also contemplated.
Additional fluorocarbons not listed here, but having the properties described in this disclosure that would lend themselves to therapeutic applications, are also contemplated. Such fluorocarbons may be commercially available or specially prepared. As will be appreciated by one skilled in the art, there exist a variety of methods for the preparation of fluorocarbons that are well known in the art. See for example, Schweighart, U.S. Pat. No. 4,895,876, which is incorporated by reference in its entirety.
2. The Secondary Fluorocarbon
The secondary fluorocarbon can be an aliphatic fluorocarbon substituted with one or more lipophilic moieties and having a higher molecular weight than the first fluorocarbon. The lipophilic moiety may be terminally substituted on the fluorocarbon molecule. Preferably, the molecular weight of the second fluorocarbon is greater than about 540 Daltons. Constraints on the upper limit of the molecular weight of the second fluorocarbon are often related to its organ retention time and its ability to be solubilized by the first fluorocarbon. Most preferred second fluorocarbons have boiling points greater than about 150° C. and water solubilities of less than about 1×10−9 moles/liter.
Of course, as will be appreciated by one skilled in the art, many fluorocarbons substituted with different lipophilic groups could be suitably used as the second fluorocarbon in the present invention. Such fluorocarbons may include esters, thioethers, and various fluorocarbon-hydrocarbon compounds, including isomers. Mixtures of two or more fluorocarbons satisfying the criteria set forth herein are also encompassed within the broad definition of fluorocarbon materials suitable for use as the second fluorocarbon of the present invention. Fluorocarbons not listed here, but having the properties described in this disclosure that would lend themselves to therapeutic applications, are additionally contemplated.
The lipophilic moiety is optimally selected from the group consisting of Br, Cl, I, CH3, or a saturated or unsaturated hydrocarbon of 2 or 3 carbon atoms. Consequently, preferred second fluorocarbons may be selected from the group of terminally substituted fluorocarbon halides as represented by the general formula:                CnF2n+1X or CnF2nX2, wherein n is 8 or greater, preferably 10 to 12, and X is a halide selected from the group consisting of Br, Cl, or I;1-alkyl-perfluorocarbons or dialkylperfluorocarbons as represented by the general formula:        CnF2n+1—(CH2)n′CH3 wherein n is 8 or greater, preferably 10 to 12, and n′ is 0 to 2;1-alkenyl-perfluorocarbons as represented by the general formula:        CnF2n+1—Cn′H(2n′-1, wherein n is 10 or more, preferably 10 to 12, and n′ is either 2 or 3; orbrominated linear or branched perfluoroethers or polyethers having the following general structure:        Br—(CnF2n+1—O—Cn′F2n+1), wherein n and n′ are each at least 2 and the sum of n and n′ is greater than or equal to 8.        
Most preferably, the second fluorocarbon of the present invention is selected from the group consisting of linear or branched brominated perfluorinated alkyl ethers, perfluorodecyl bromide (C10F21Br); perfluorododecyl bromide (C12F25Br); 1-perfluorodecylethene (C10F21CH═CH2); and 1-perfluorodecylethane (C10F21CH2CH3); with perfluorodecyl bromide particularly preferred.
The question that must be asked of the particular secondary fluorocarbon is whether the increased stability provided by the secondary fluorocarbon is outweighed by the potential problems of prolonged organ retention time, and half-life, toxicity, biocompatibility and other complications.
The chemical formula of perfluorooctyl bromide (“PFOB”) is C8F17Br. Its structure is CF3(CF2)6CF2Br and the Chemical Abstract Service (“CAS”) number is 423-55-2. Other physical parameters of PFOB include:
Boiling point143° C. at 760 mm HgVapor pressure10.5 mm Hg at 37° C.Melting point4° C.
The chemical formula of PFDB is C10F21Br. Its structure is CF3(CF2)8CF2Br. The CAS number is 307-43-7. Other physical parameters include:
Boiling point180° C. at 760 mm HgVapor Pressure1.5 mm Hg at 37° C.Melting point55° C.
The addition of perfluorodecyl bromide (“PFDB”) to perfluorooctyl bromide (“PFOB”) or perfluorodecalin (“FDC”) emulsions has been found to result in excellent room temperature stability. Due to its lipophilic character, the half-life of PFDB in the RES is only 23 days, a value deemed acceptable for intravenous applications. The addition of PFDB is also found to result in narrow particle size distributions and fewer large particles. With the addition of PFDB to a PFOB emulsion, significant partitioning of the two components between different droplet sized droplets occurs as a consequence of molecular diffusion, whereby the insoluble component becomes concentrated in the smaller droplets, and the high solubility component enriches the larger droplets. The partitioning of the two components between different sized droplets leads to decreased solubility for the smaller droplets via Raoult's Law. This compensates for the difference in chemical potentials caused by differences in capillary pressures (i.e. the Kelvin effect). When the concentration balances the capillary effect, droplet growth ceases.
The emulsion growth rate {acute over (ω)}ab, for a two component disperse phase is given by the following equation:{acute over (ω)}ab=1/[(Φa/{acute over (ω)}a)+(Φb/{acute over (ω)}b)]where Φa and Φb are the volume fractions of fluorocarbons a and b, respectively. {acute over (ω)}a and {acute over (ω)}b are the individual emulsion growth rates given by the above equation for fluorocarbons a and b.
While PFDB has proven to be an ideal candidate for a secondary fluorocarbon, PFDB should be formulated in amounts, with the appropriate primary fluorocarbon (e.g., PFOB) at appropriate concentrations, so as not to cause complications with organ retention and half-life, but still provide proper emulsion stability. Further, because there is a desire to use any fluorocarbon emulsion repeatedly on patients in as short time intervals as possible, the smallest amounts of PFDB should be used to allow for repeat doses. This should be in combination with the appropriate amount of primary fluorocarbon.
A further complication of PFDB being present in the emulsion in too high of a concentration is the significant risk of PFDB crystals forming in the emulsion due to its high melting point (55° C.). Crystallization of PFDB in the emulsion could cause serious safety and toxicity issues. There is also a significant manufacturing concern in that high concentrations of PFDB can easily crystallize when encountering cold spots in the manufacturing process. This is a serious consideration for formulating an emulsion, particularly at large scale manufacturing, in that should PFDB crystallize during the manufacturing process, because it is the emulsion stabilizer, it would not only present biocompatibility/toxicity issues but also its crystallization would make less PFDB available to stabilize the emulsion and the finished emulsion would be much less likely to meet product quality specifications. Another complication in PFDB being present in too high of a concentration is the risk of making particles too small, which can cause fluorocarbon particles to leak from the capillary beds into the interstitial space of tissues causing severe retention problems.
Thus, creating a usable fluorocarbon emulsion for in vivo use in humans requires the balancing of several critical considerations. A fluorocarbon emulsion designed for in vivo oxygen transport in humans should have at least the following attributes:
1. Acceptable organ half-life—The organ half-life of the fluorocarbon emulsion components should be as short as possible, preferably less than 4 weeks.
2. Acceptable shelf stability—The fluorocarbon emulsion should have a minimum of 6 months storage at 5° C., with 18 months of shelf life preferred. During the storage period, the physical characteristics and biocompatibility of the fluorocarbon emulsion should not change. Room temperature storage is preferred because of the added convenience, greater potential application and reduced expense;3. Acceptable in vivo stability—The emulsion should not undergo phase changes, precipitation, coacervation, coalescence or other aggregative phenomena of an adverse physicochemical or biochemical nature when administered intravenously;4. Excellent biocompatibility—The fluorocarbon emulsion should induce minimal side-effects and no toxic effects;5. Acceptable or optimal particle size and distribution—The emulsion particle size plays an important role both due to size and distribution in toxicity, shelf life, side effects, and biodistribution and should therefore be within acceptable ranges;6. Terminal sterilization—The emulsion should be of a physical characteristic to be able to be terminally sterilized;7. Acceptable blood half-life—The emulsion should have a blood half-life which is acceptable for its intended purpose;8. Acceptable viscosity—The viscosity of the whole blood/fluorocarbon emulsion mixtures is critical since tissue oxygenation and perfusion are inversely related to viscosity;9. Low free fluoride values—The fluorocarbon should exhibit excellent chemical stability;10. Surfactant safety—The surfactant should be biocompatible and non-toxic;11. Acceptable fluorocarbon content—The biocompatibility and toxicity of a fluorocarbon depends critically on the total fluorocarbon content and must be efficacious in volumes acceptable to sick patients and should be formulated for repeat doses;12. Manufacturing—The fluorocarbon emulsion should have physical properties which allow it to be easily manufactured and on a scaled up basis to produce consistent product. The fluorocarbons used in the formulation should be easily produced and with excellent purity; and,13. Repeated dosage—The fluorocarbon emulsion should be able to be used repeatedly in as short of time intervals as possible for repeated dosing of patients.